One of the most important steps in pathogenesis of infections is the attachment of microbes to host tissues. Pathogenic bacteria bind to target cells via specific adhesive organelles. Over 30 different operons, encoding virulence associated surface structures of pathogenic Gram-negative bacteria, such as Escherichia coli, Haemophilus influenzae, Salmonella enteriditis, Salmonella typhimurium, Bordetella pertussis, Yersinia enterocolitica, Yersinia pestis, Helicobacter pyroli and Kebsiella pneumoniae been identified as members of a particular family using the chaperone/usher protein assisted assembly pathway (Thanassi, D. G. et al., 1998, Curr. Opin. Microbiol. 1, pp. 223-231; Soto, G. E. and Hultgren, S. J. ,1999, J. Bacteriol. 181, pp. 1059-1071).
In contrast to the apparent complexity of the general secretory (Type II; Karlyshev, A. V. and MacIntyre, S., 1995, Gene 158, pp. 77-82) and contact mediated (Type III; Lory, S., 1998, Curr. Opin. Microbiol. 1, pp. 27-35) pathways the chaperone/usher pathway appears to be rather simple and specific for secreting structural subunits. In addition to the structural subunits, these latter operons encode only two proteins involved in export and assembly. One is a periplasmic chaperone, which shows specificity for the structural subunit(s). The other one is a large outer membrane usher protein, which is required for translocation across the outer membrane, and which may form a large gated channel. The prototype of this pathway has been the PapD chaperone/PapC usher mediated assembly of Pap pili of Escherichia coli uropathogenic strain (Thanassi, D. G. et al., 1998, Curr. Opin. Microbiol. 1, pp. 223-231). The 3D-dimensional structure of PapD has been solved (Holmgren, A. and Branden, C. I., 1989, Nature 342, pp. 248-251). It has two domains, each with β-barrel and an immunoglobulin (Ig)-like fold.
Based on conserved differences in the primary structure of periplasmic chaperones and of organelle subunits, chaperone/usher systems may be divided into two subfamilies (denoted FGS and FGL) (Zav'yalov, V. P. et al., 1995, FEMS Immunol. Med. Microbiol. 11, pp. 19-24; Hung, D. L. et al., 1996, EMBO J. 15, pp. 3792-3805) involved in the assembly of morphologically distinct surface structures. FGS chaperone/usher systems are used for assembly of rod-like organelles (e.g. P and type 1 pili in E. coli), whereas thin aggregative fibrillar adhesins (TAFA) are assembled by FGL systems.
FGL chaperones are characterized by an extended variable sequence between the F1 and G1 β-strands, a disulfide bond connecting these two strands, and an extended N-terminal sequence (Zav'yalov, V. et al., 1997, Biochem. J. 324, pp. 571-578; Chapman, D. A et al., 1999, J Bacteriol. 181, pp. 2422-2429.
The crystal structures of the P pilus PapD-PapK chaperone-adapter subunit complex (Sauer, F. G. et al., 1999, Science 285, pp. 1058-1061) and the type 1 pilus FimC-FimH chaperone-adhesin complex (Choudhury, D. et al., 1999, Science 285, pp. 1061-1066) have revealed that pilin subunits, just as the chaperones, have Ig-like folds. However, the final (seventh) β-strand of the fold is missing, creating a deep cleft on the surface of the subunit where part of the hydrophobic core is exposed. The chaperone binds to the pilin domain by donating its G1 β-strand to complete the pilin Ig-like fold (Sauer, F. G. et al., 1999, Science 285, pp. 1058-1061; Choudhury, D. et al., 1999, Science 285, pp. 1061-1066). The chaperone G1 β-strand is inserted into the pilin acceptor cleft with extensive main-chain-to-main-chain hydrogen bonding between the donor strand and the two pilin edge strands (A and F) that define the perimeters of the acceptor cleft. Alternating hydrophobic side chains in the G1 strand bind in sub-pockets within the acceptor cleft and complete the hydrophobic core of the pilin domain. Assembly of subunits is thought to proceed by a donor strand exchange mechanism in which the chaperone G1 donor strand interaction is replaced by a similar interaction between subunits (Sauer, F. G. et al., 1999, Science 285, pp. 1058-1061; Choudhury, D. et al., 1999, Science 285, pp. 1061-1066). Indeed, the X-ray analysis of PapD-PapK complex showed that N-terminal sequence of the subunit composed of 10 residues is disordered in structure (Sauer, F. G. et al., 1999, Science 285, pp. 1058-1061). This sequence possesses highly conserved motif, which have been shown to participate in subunit-subunit interactions. This motif has homology to G1 β-strand of the chaperone (Soto, G. E. et al., 1998, EMBO J. 17, pp. 6155-6167). Because the N-terminal sequence apparently protrudes away from the main body of the chaperone-subunit complex, it would be free to exchange with G1 strand of the chaperone.
Yersinia pestis induced plague was one of the most devastating diseases of the last millennium. Over a third of the population of Europe is estimated to have died in the fourteenth century epidemic. Today, the World Health Organization reports 1,000 to 3,000 cases of plague every year. One of the Y. pestis-specific properties is the capability to form capsule. The ability to elaborate capsular material (fraction 1; F1) is associated with the resistance of bacteria to phagocytosis. Absence of only capsular F1 (Caf1) antigen synthesis in the infected strain leads to increased survival time of some animal host species (Drozdov, I. G. et al., 1995, J. Med. Microbiol. 42, pp. 264-268; Samoilova, S. V. et al., 1996, J. Med. Microbiol. 45, 440-444).
The nonpilus organelles (e.g. F1 capsular antigen and Dr adhesins) are composed of only one or sometimes two types of subunits. The F1 capsular antigen from the plaque pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and it serves as a prototype for nonpilus organelles assembled via the FGL chaperone/usher pathway. Therefore, while the F1 capsular antigen from Yersinia is used as the model in the present disclosure, the invention is related also to other microbes using the same assembly pathway. Urinary track infection (UTI) caused by Eschericia coli for example, is a severe health problem all over the world. Alone in the USA approximately 1 million patients are hospitalised each year due to this infectious disease.
Based on the particular properties of the primary structure of periplasmic chaperone, the caf operon, responsible for production and assembly of Caf1 antigen (Galyov, E. E. et al., 1990, FEBS Lett. 277, pp. 230-232), is related to FGL subfamily of chaperone/usher systems. It encodes a 26.5 kDa periplasmic chaperone Caf1M (Galyov, E. E. et al., 1991, FEBS Lett. 286, pp. 79-82) and a 90.4 kDa outer membrane usher protein Caf1A (Karlyshev, A. V. et al., 1992, FEBS Lett. 297, pp. 77-80) which together mediate surface assembly of Caf1 subunits in recombinant E. coli (Karlyshev, A. V. et al., 1994, NATO ASI Series, v. 11, H 82, Biological Membranes: Structure, Biogenesis and Dynamics, pp. 321-330).
Accordingly, preventing the formation of adhesive organelles is therefore a promising concept to prevent the bacteria to infect host tissues. There are disclosures showing various potential compounds to prevent the formation of adhesive organelles. However, basically these inventions disclose clearly more complex compounds than the present disclosure. Moreover, the prior art concentrates in the peptides of the G1 β-strand as being potential antimicrobials.
U.S. patent application 0030099665 discloses a vaccine against bacterial infections comprising a complex of a bacterial chaperone protein with an adhesin protein or an mannose binding immunogenic fragment of the adhesin protein. The vaccine is especially for treating of urinary tract infection.
U.S. patent application Ser. No. 20020086037 discloses a protein construct comprising a pilus protein portion an a donor strand complementary segment.
U.S. Pat. No. 6,001,823 discloses a method to screen for drugs against diseases caused by tissue-adhering bacteria. The method is based predicting the binding energy of a putative drug molecule to the chaperone.
In this invention surprisingly we found that only short peptide sequences (about 6 amino acid residues) at the N-terminal extension of Caf1 subunit are critical for Caf1 polymerization and expression on bacterial surfaces. Surprisingly, we additionally observed that peptides corresponding to the sequences effectively inhibited surface expression of Caf1.